It is proposed to develop new instrumentation and methodology which can be used for sequencing peptides and post-translationally modified peptides at the low picomole level and below. This work will be developed specifically for sequencing tumor related proteins isolated using 2-D Gel electrophoresis in the 2-D Gel Laboratory at the University of Michigan. These proteins are enzymatically cleaved into peptide fractions and the resulting peptides often can not be readily sequenced by the Edman method due to either N-terminal blockage, low amounts of sample, or the inability to detect post-translational modifications. This proposal will thus involve further development of an ion trap/reflectron time-of- flight mass spectrometer and the development of various capabilities that result from the combination of this hybrid instrument. This device will be used to obtain sequence information on peptide digests using matrix-assisted laser desorption (MALDI) for producing large ions in the trap directly or by using electrospray ionization with external injection into the trap. The ion trap will act as a front end storage device for a time-of-flight mass spectrometer with capabilities for long term storage, thus providing enhanced sensitivity for detection of the ultra low levels of peptides obtained from 2-D Gels. The storage of the IT/re TOF will also provide the ability to investigate photodissociation and collision induced dissociation with MS/MS capabilities.The long term storage of large ions in the trap will be shown to be especially important for detection of long-lived unimolecular fragmentation of these large species, which has been shown to occur on a time scale beyond the capabilities of most mass spectrometers. The various factors that allow sufficient fragmentation for interpretation of the structure of peptides in this process will be investigated. In addition, the trapping capabilities of the IT/reTOF will provide sufficient resolution to resolve the isotopic distribution of the molecular ions of peptides and of their fragment peaks so that different fragments obtained from electrospray ionization can be identified according to their charge states. Ultimately, through using fragmentation patterns observed with a reflectron time-of-flight device, a demonstration of the ability to obtain structural analysis will be presented. In addition, various methods for on-line introduction of separated peptide fractions will be investigated using the enhanced resolution and speed of the IT/reTOF.